Hepatitis C is the main cause of hepatitis acquired by transfusion. Hepatitis C may also be transmitted by other percutaneous routes, for example by intravenous drug injection. The risk of contamination of health professionals is moreover not negligible.
Hepatitis C is distinguishable from other forms of liver diseases associated with viruses, such as hepatitis A, B or D. Hepatitis C virus (HCV) infections are often chronic with, as a consequence, liver diseases such as hepatitis, cirrhosis and carcinoma in a large number of cases.
Although the risk of transmission of the virus through transfusion has diminished because of the selection of blood donors, the frequency of hepatitis C remains high. Currently, about 170 million people worldwide are chronically infected with HCV. Populations at high risk are found mainly in recipients of blood transfusions and intravenous drug users, but asymptomatic blood donors exist who do not belong to these high-risk groups and in whom circulating anti-HCV antibodies have been found. For the latter, the route of infection has not yet been identified.
HCV was the first hepatotropic virus isolated by means of molecular biology techniques. The sequences of the viral genome were cloned before the viral particle was visualized.
HCV is a 9.5 kb, positive single-stranded RNA virus which replicates via a complementary RNA copy and whose translational product is a precursor of a single polyprotein of about 3000 amino acids. The 5xe2x80x2 end of the HCV genome corresponds to an untranslated region adjacent to the genes which encode the structural proteins, the core protein of the nucleocapsid and the two envelope glycoproteins, E1 and E2/NS1. The untranslated 5xe2x80x2 region and the core gene are relatively well conserved in the various genotypes, but the E2 envelope proteins are encoded by a hypervariable region which is different from one isolate to another. The 3xe2x80x2 end of the HCV genome contains the genes which encode the nonstructural (NS) proteins and a well conserved noncoding 3xe2x80x2 region.
Because of its genomic organization and its presumed mode of replication, HCV has been classified in a novel genus of the Flaviviridae family, the Hepaciviruses.
Numerous techniques have been developed for the diagnosis of an HCV infection. For example, diagnostic immunological assays have been carried out to detect antibodies directed against HCV proteins in the sera of patients. The synthesis of cDNA by reverse transcription of the viral RNA and PCR amplification have also been used to detect the HCV genome, as the indirect measurement of a potentially infectious virus in the sera of chronically infected humans or those of experimentally infected chimpanzees. Moreover, on the basis of gene cloning, hybridization techniques with a DNA probe have also been developed.
However, it is recognized that existing diagnostic techniques lack sensitivity and/or specificity and/or suffer from implementation difficulties. By way of example, with the method of hybridization of probes, it is impossible to distinguish between a virus with low infectivity and a virus with high infectivity. It is therefore necessary, but difficult to carry out, to inoculate the virus which has to be tested into a chimpanzee and to test the resulting infection on the animal.
It is therefore of primary importance, from the point of view of public health, to be able to develop specific, sensitive and practical methods for identifying and screening HCV carriers. One of the solutions could be to produce a very efficient in vitro system for culturing HCV which would make it possible to obtain propagation of the virus, in particular to study its mechanisms of replication, to test neutralizing antibodies or antiviral agents, as well as to develop biological materials, diagnostic trials and vaccine preparations. Indeed, although the complete HCV sequence has been available since 1989 (Q. L Choo et al., Science 244, 359 (1989)), understanding of the life cycle and the mode of replication of HCV has been hampered by a lack of an appropriate in vitro culture system. Ito et al. (J. Gen, Virol. 77: 1043-1054 (1996)) have indeed confirmed maintenance of the replication of HCV in primary cultures of human hepatocytes obtained from patients carrying HCV and for whom the disease was chronically established, and suggested a passage of infection, but problems relating to the propagation of the virus remain (impossibility of long term culture) and the system developed is limited by the need for a supply of human liver and the cumbersome nature of the technique. Moreover, up until now, there is no general consensus on the tropism of HCV and all the cellular receptors for the virus have not yet been identified.
Viral RNA-containing particles which are very heterogeneous in density are present in the plasma of patients infected with HCV. This heterogeneity in the density of the particles containing viral RNA is attributed to their association in variable proportion with lipoproteins (Thomsen et al., 1993, Med. Microbiol. Immunol. 182: 639). In the description of the present patent application, the inventors have called these hybrid particles LVPs (lipo-viro-particles). The distribution of each of these forms along a density gradient varies from one patient to another. The existing analyses of particles of low density show densities covering those of the LDLs (Low Density Lipoproteins) and of the VLDLs (Very Low Density Lipoproteins). The size described (50 nm) bring them close to the VLDLs.
The nature of the abovementioned LVPs containing viral RNA is up until now not precisely known, but the present inventors have shown for the first time that the LVPs are associated with human immunoglobulins and that it is in these fractions of LVPs, whose density is equal to or less than 1.063 g/ml, associated with human immunoglobulins, that the HCV virus RNA is predominantly present, contrary to the known data (Hijikata et al., J. Virol. (1993), 1953-1958). Indeed, Hijikata et al. have shown that there were no human immunoglobulins in the particles having a density of less than 1.06 g/ml, and that it is in the particles of this density that a high infectivity is found in chimpanzees. These experimental data could constitute at least one basis of explanation of the chronicity of the disease, which has not yet been elucidated up until now, and furthermore open perspectives for novel methods of in vitro culture of the HCV virus.
Accordingly, the subject of the present invention is a complex consisting of LVPs associated with human immunoglobulins, the said complex having a density of less than 1.063 g/ml and preferably between 1.0063 and 1.063 g/ml, as demonstrated by centrifugation, for example on a sodium bromide gradient.